Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. SMA is caused by reduced levels of the survival motor neuron (SMN) protein - reflecting homozygous loss of the SMN1 gene but preservation of the nearly-identical SMN2 gene. Low SMN levels lead to motor neuron degeneration and loss of muscle strength. There is therefore an urgent need to identify treatments that can restore SMN levels or can correct the deficits downstream of SMN depletion. Since SMA patients and mouse models with higher copy numbers of the SMN2 gene show reduced disease severity, most drug screening efforts have focused on enhancing expression of full-length SMN from the SMN2 gene. This may occur either by increasing SMN2 transcription or by correcting the splicing defect, which leads it to be hypomorphic. However, most of the existing screening assays do not directly assess SMN function but instead measure reporter gene activity and/or SMN protein accumulation. Therefore, compounds that have a small effect on overall levels but a significant effect on SMN function may go undetected. Moreover, compounds acting on the disease pathway rather than on the disease trigger are not screened for. The current proposal aims to address each of these shortcomings. We have generated mouse fibroblast lines with regulated knockdown of endogenous SMN. Reducing mSmn expression to a level similar to that found in tissues of type I patients triggers growth arrest in these cells - a phenotype that can be corrected by ectopic expression of RNAi-resistant human SMN. In support of the idea that this assay mirrors the mechanisms at play in the disease, the ability of different point mutant forms of SMN to correct the growth defect is proportional to their potency in rescuing the motor phenotype in mouse models. We will develop a modified version of this cell line in which cell proliferation is dependent on SMN levels produced by the human SMN2 gene. The untreated cell line will show reduced growth, while agents that enhance SMN2 transcription or splicing, stabilize SMN protein, or correct downstream defects will promote increased proliferation. This new model will therefore provide a functional readout for multiple potential therapeutic targets in SMA. Following initial testing of the assay for reproducibility and responsiveness to known benchmarking compounds, we will perform a pilot screen of 4,000 chemical compounds known to show biological activity. Secondary screens have been devised to eliminate compounds acting through mechanisms that are not disease-related and to distinguish between those acting at the level of SMN2 and others acting downstream. The screen is designed to provide a first insight into mechanism(s) of action and priority ranking of compounds for follow-up studies. These will be of three types: i) partnerships with academic and pharmaceutical screening centers to pursue a large-scale high-throughput screen on the validated assay, ii) analysis of potential disease mechanisms indicated by the hit compounds, and iii) investigation of therapeutic potential of the hit compounds in mouse models of SMA. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA) is the most common genetic cause of death in infancy, but no effective treatment is available. We are developing new cell models which allow us to mimic the defect in the culture dish. As a first step toward new therapeutic strategies, we will test thousands of chemical compounds for their ability to correct the SMA-related defect in these cells.